Two novel extracellular cholesterol oxidases designated CO1 and CO2, from Bacillus sp. SFF34, were purified 5.6 and 5.9-fold giving Mr values of 36 and 37 kDa. The optimum temperature for the activity was 60 degreesC (CO1) and 40 degreesC (CO2), and the optimum pH was 6.25 (CO1) and 6 (CO2) over 30 min reaction time. The apparent K-m values for cholesterol were 6.76 mM (CO1) and 4.50 mM (CO2). Both the enzymes could oxidize 5alpha-cholestane, 5alpha-cholestane-3beta-ol-7-one, coprostane, dihydrocholesterol, hecogenin, beta-sitosterol and stigmasterol.